Supercritical fluids processing: preparation of protein microparticles and their stabilisation

ABSTRACT

A coprecipitate of protein or polypeptide and stabiliser obtained by a process for the co-precipitation of a substance with a stabiliser therefor, by a gas anti solvent process comprising introducing into a particle formation vessel a supercritical fluid pure or mixed with a modifier; and a solution comprising said substance and said stabiliser dissolved in a solvent; so as said solvent is extracted from the solution by said supercritical fluid and co-precipitation of the substance and stabiliser occurs. The process may be carried out using an apparatus comprising a particle formation vessel and a nozzle having a central orifice serving to introduce a solution of the substance and a plurality of outer orifices serving to carry a flow of supercritical fluid into the particle formation vessel, such that the solvent is extracted from the solution by the supercritical fluid and precipitation of micron sized particles of the substance/stabiliser occurs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims the benefit of, U.S.application Ser. No. 10/493,256, filed Sep. 13, 2004; which is anational stage application of PCT/EP02/11761, filed Oct. 21, 2002; whichclaims priority to European Application No. 01125048.7, filed Oct. 22,2001; all of which are hereby incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to a method for protein and polypeptideprecipitation by supercritical fluid processing and their protection andstabilisation against denaturation.

BACKGROUND OF THE INVENTION

The need for stable proteins and polypeptides for many applications iscontinuously increasing. This is particularly pronounced for therapeuticproteins in the pharmaceutical field. For the ease of both manufacturersand final users aqueous protein solutions are often the preferred formof administration. Moreover this is their common natural form thatallows hydrated, three-dimensional folded complex formation. Thisconformation is generally reported as tertiary structure and itsintegrity is of vital importance for maintaining the biological activityof proteins. The irreversible loss of tertiary structure of proteins isreferred as denaturation and causes inactivation. Because proteins andpolypeptides in solution are exposed to many stresses which can causephysical (denaturation) and chemical (i.e., reactions such ashydrolysis, deamidation etc . . . ) degradation, very often thedevelopment of liquid formulations is precluded. Presently, the mostcommon way to achieve protein stability is the removal of water bysuitable processes such as freeze-drying or spray-drying. However, bothof these techniques (ref. “Formulation and Delivery of Proteins andPeptides” J. L. Cleland and R. Langer, American Chemical Society,Washington, D.C. 1994) can induce protein unfolding. In particular withregard to lyophilisation protein unfolding can occur either during theinitial freezing step or during acute dehydration by sublimation.

Concerning spray drying, thermal degradation, low efficiency, low yieldand high levels of residual moisture are the main limitations of thetechnique.

Another problem is the difference in long term stability of analogousformulations obtained by different drying processes. In fact, dependingon the dehydration method, the protein may assume differentthree-dimensional structures with the same initial biological activitybut different shelf life.

The stabilising effect of carbohydrates and in particular of trehaloseon proteins during freezing and dehydration is well documented(“Formulation and Delivery of Proteins and Peptides” J. L. Cleland andR. Langer, American Chemical Society, Washington, D.C. 1994 and“FreezeDrying/Lyophilization of Pharmaceutical and Biological Products”L. Rey and J. C. May, Marcel Dekker, Inc. New York 1999). Although manysugars can prevent protein damage during dehydration, the products oftenhave a short shelf life at room temperature due to the Maillardreaction. Stability at room temperature can be improved using nonreducing sugars such as sucrose and trehalose.

British Patent Application GB 2009198 discloses lyophilisation ofmeningococcal polysaccharide and trehalose; GB 2126588 discloses thestabilisation of tumour necrosis, factor (TNF) to lyophilisation andfreezing by including either a non-ionic surfactant or trehalose (oranother sugar); and Japanese Patent Application J 58074696 discloses thefreeze-drying of ATP in presence of trehalose.

Preparations of alkaline phosphatase containing trehalose are reportedto maintain their activity after freeze-drying and to maintain about 70%of the initial activity after 84 days/storage at 45° C. (A. W. Ford etAl., J. Pharm. Pharmacol. 1993, 45: 86-93). Although lyophilisation isstill the main process used for drying proteins, several precautionsmust be taken in order to avoid the damage that severe stressing phasessuch as freeze-thawing and drying can cause. In fact, during the firststep in freeze-dried protein formulation, a correct choice of conditions(pH, ionic strength, presence of stabilisers, etc . . . ) guarantees thebest protection against protein unfolding and inactivation. Manyexcipients such as sugars, aminoacids, polymers, surfactants specificligands (substrates, co-factors, allosteric modifiers etc . . . ) areknown to stabilise proteins during freeze-drying and have been named“lyoprotectants.” Among them, carbohydrates and in particulardisaccharides such as sucrose and trehalose have been widely studied.The stabilising mechanism of these compounds as well as otherstabilisers has not been completely clarified. However, an effectivelyoprotectant must maintain stability during both freeze-thawing anddrying. Since the protein environment is aqueous during much of thefreezing process, solutes that stabilise the native conformation inaqueous solutions are very often effective as protein cryoprotectants.Carbohydrates and some aminoacids are examples. Arakawa et al. (J.Pharm. Res. 1991, 8, 285-29 1) reported that such solutes tend to beexcluded from the surface of protein while in aqueous solution. Thethermodynamic consequence of such phenomenon is the stabilisation ofprotein native conformation.

Stability during drying and storage is best explained by both the watersubstitution and the vitrification hypotheses. The first states thatstabilisers interact with the protein as water does by replacing theremoved water and accounts for the thermodynamic control of dryingprocess. The latter states that stabilisers are good glass formers andremain amorphous during and after drying so that they mechanicallyimmobilise proteins inside a glassy matrix. This is a purely kineticargument that applies equally well to both drying and storage stability.(“Freeze-Drying/Lyophilization of Pharmaceutical and BiologicalProducts” L. Rey and J. C. May, Marcel Dekker, Inc. New York 1999).

Hence, referring to the above stabilisation hypothesis of driedproteins, it can be postulated that vitrification is one of the mainissues for long term stability. The use of spray-drying for proteindesiccation has been less investigated. Although fine amorphousparticles can be produced, this process requires warm air as a dryingforce that can lead to protein thermal degradation. Moreover, lowefficiency, low yield and high levels of residual moisture are otherlimitations.

Another reported technique for drying proteins which should avoidinactivation is air dehydration at room temperature. U.S. Pat. No.4,891,319 of Quadrant Bioresources Ltd (UK) discloses the preservationof several proteins and other macromolecules at 37-40° C. by drying inpresence of trehalose at atmospheric pressure.

The use of supercritical fluid technology has also been reported as auseful method for obtaining proteins as dry fine micro-particles. Themain advantages of this technique are the possibility of maintaining theprotein in a favourable aqueous environment before a rapid precipitationin order to minimise denaturation and the process length which isshorter than freeze-drying and less expensive.

S. P. Sellers et al. (J. Pharm. Sci., 2001, 90, 785-797) report adehydration method for protein powder production based on supercriticalCO₂-assisted nebulization. This technique can be assimilated tospray-drying; in fact supercritical CO₂ is used for enhancing solutionnebulization and not as an anti-solvent solvent for soluteprecipitation. The GAS (Gas Anti-Solvent recrystallization) process toform protein microparticles is reported by Debenedetti (U.S. Pat. No.6,063,910). In this case the protein solution is sprayed through a laserdrilled platinum disc with a diameter of 20/inn and a length of 240/inninside the particle formation vessel previously filled by supercriticalfluid which is introduced by a different inlet. This technique has beenused to form particles of catalase and insulin (0.01% w/v) fromethanol/water (9:1 v/v) solutions using carbon dioxide as thesupercritical fluid. In this process, the supercritical fluid inlet isnot optimized: the solution injection occurs in an almost staticatmosphere of supercritical fluid, with low turbulence. Hanna M. andYork P. (W096/00610) proposed a new method and a new apparatus to obtainvery small particles by a specific supercritical fluid technique namedSEDS (Solution Enhanced Dispersion by Supercritical Solution).

The process is based on a new coaxial nozzle: the solution expandsthrough a capillary inlet, supercritical fluid expands through anexternal coaxial pathway with a conical shaped end. The mixing betweensupercritical fluid and solution occurs in the conical zone. They alsopropose the use of a three way nozzle: a modifier can be fed in order toimprove the mixing.

They applied the SEDS technology to precipitation of small particles ofwater soluble compounds, such as sugars (Lactose, Maltose, Trehalose andSucrose) and proteins (R-TEM beta-lactamase). Co-precipitation ofproteins and stabilisers is not mentioned nor exemplified therein.

Moreover, the same inventors (WO001/03821) describe an improvedprecipitation method using the same apparatus but feeding to theparticle formation vessel a supercritical fluid and two immisciblesolvents. This method allows co-precipitation of two or more solutesdissolved in the two immiscible solvents. The fluids inlet is formed bya coaxial nozzle wherein contact between the two solvents occurs shortlybefore their dispersion by the supercritical anti-solvent, avoiding theprecipitation solutes inside the nozzle. However this method permits theformation of homogeneous co-precipitates; it is generally useful whentwo solutes with different polarity must be processed. Moreover, if thisis used for an aqueous solution, the second solvent must be at leastpartly soluble in water so that it allows the water to disperse in thesupercritical anti-solvent. This step is necessary to permitwater-soluble solute precipitation. Co-precipitation of proteins andstabilisers is not described in this document.

Walker (W00/15664) discloses a method for co-formulating an active(preferably a pharmaceutically active) substance and an oligomeric orpolymeric excipient in which an amount between 80 and 100% of the activesubstance is in amorphous as opposed to crystalline form. In theseformulations the active substances are more stable compared to thecrystalline forms when stored at temperature between 0 and 10° C. Onlythe co-formulation of a pharmaceutical active substance with anoligomeric or polymeric excipient is disclosed and there is no mentionof protein stabilisation in this document. Protein stabilisation istherefore achieved in the art through freeze-drying and spray-drying.The co-precipitation of proteins with stabilisers using supercriticalfluids has not been described before and it is the object of the presentinvention.

We have now found a method of producing stable dry proteinmicroparticles by co-precipitation with a stabiliser using supercriticalfluids. Preferred stabilisers are carbohydrates, aminoacids, surfactantsand polymers. More preferably the stabilizer is a sugar, most preferablytrehalose.

Co-precipitation allows intimate interactions between theprotein/stabiliser molecules and an optimal weight/weight ratio existsfor each couple protein/stabiliser.

In fact since there is no freeze-thawing there is no need forcryoprotection. Moreover, although the nature of protein/stabiliserinteractions has to be better clarified, in the present case thestabiliser plays the essential role of improving storage stabilityrather than that of retaining protein activity during drying. In fact,precipitation by a supercritical fluid allows by itself protein particleproduction without denaturation during the drying process.

STATEMENT OF THE INVENTION

The term “supercritical fluid” means a fluid at or above its criticalpressure and its critical temperature.

The term “solvent” means a liquid, which is able to form a solution withthe protein and the stabiliser.

The term “stabiliser” means a solid pharmaceutical excipient which isable to stabilise, for example, proteins, which is soluble in thesolvent and which is substantially insoluble in the supercritical fluid.

The term “modifier” is a substance, preferably a solvent, which enhancesthe solubility of the “solvent” in the supercritical fluid.

The present invention provides a process for the co-precipitation of asubstance with a stabiliser therefor, by a gas anti solvent processcomprising introducing into a particle formation vessel a supercriticalfluid pure or mixed with a modifier; and a solution comprising saidsubstance and said stabiliser dissolved in a solvent; so as said solventis extracted from the solution by said supercritical fluid andco-precipitation of the substance and stabiliser occurs.

Preferably, the solution is introduced into the particle formationvessel mixed with a modifier. The process includes the introduction intoa particle formation vessel of a solution or suspension of the substanceand the stabiliser and a supercritical fluid. In the particle formationvessel, mixing of the supercritical fluid with the solution andextraction of the solvent by the supercritical fluid occurs so that thesolutes (substance and stabiliser) co-precipitate as fine particles. Ifthe solvent is not miscible with the supercritical fluid, the use of amodifier is needed. The modifier is a compound which is soluble both inthe solvent and in the supercritical fluid.

More preferably, the apparatus in FIG. 1 is used. In this case, thesolution of substance and stabiliser, the supercritical fluid and themodifier, if needed, are separately introduced into the particleformation vessel in co-current flow by the nozzle 27. Such a nozzleW002/68107, which is shown in FIGS. 2 and 3, provides separate inletsfor supercritical fluid and solution. In fact, this is a disk with anorifice at its center and two or more orifices at the same distance fromthe center and evenly spaced along a circumference. All the orificescommunicate with the interior of the particle formation vessel. Thesolution is introduced into the particle formation vessel through thecentral orifice, while the supercritical fluid, pure or with themodifier, is introduced through the outer orifices.

The modifier and the supercritical fluid are mixed before theintroduction into the particle formation vessel. In another version ofthe process the modifier is introduced into the particle formationvessel in part with the solution and in part with the supercriticalfluid or with the solution only.

The substance is preferably a protein or polypeptide compound ofpharmaceutical or diagnostic interest, soluble in the solvent and in themixture solvent/modifier and substantially insoluble in thesupercritical fluid.

The stabiliser is preferably a pharmaceutical excipient which is able tostabilise the substance in the co-precipitated product. The stabiliseris soluble in the solvent and in the mixture solvent/modifier andsubstantially insoluble in the supercritical fluid. Preferably, thestabilizer is a sugar and more preferably is trehalose. A mixture ofstabilizers may also be employed.

The solvent is preferably selected from water, ethanol, methanol, DMSO,isopropanol, acetone, THF, acetic acid, ethyleneglycol,polyethyleneglycol and N,N-dimethylaniline. Most preferably the solventis water.

The supercritical fluid is preferably selected from carbon dioxide,ethane, ethylene, propane, sulfur hexafluoride, nitrous oxide,chlorotrifluoromethane, monofluoromethane, xenon and their mixtures,most preferably carbon dioxide.

The modifier is preferably selected from ethanol, methanol, DMSO,isopropanol, acetone, THF, acetic acid, ethyleneglycol,polyethyleneglycol and N,N-dimethylaniline or mixtures thereof. Mostpreferably the modifier is ethanol. The modifier and solvent must ofcourse be different.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic flow sheet of the apparatus used to carry outthe process according to this invention.

FIGS. 2 and 3 show the nozzle that is used to carry out the processaccording to this invention.

FIGS. 4, 5 and 6 show the particle size distributions of supercriticalCO₂ co-precipitated lysozyme/trehalose powders at the w/w ratios 1:10,1:2 and 1:0 respectively.

FIG. 7 shows the thermograms obtained by differential scanningcalorimetry (DSC) of supercritical CO₂ co-precipitatedlysozyme/trehalose powders vs the respective pure products.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be described further with particular reference to thesubstance being a protein. It has been found that it is possible toproduce stable dry protein/stabilizer micro-particles through the use ofsupercritical fluids, using various stabilisers such as carbohydrates,aminoacids and surfactant polymers.

Surprisingly, it has been found that co-precipitation usingsupercritical fluids allows particular intimate interactions betweenprotein and stabiliser molecules and that for each protein/stabilisercouple there is an optimal weight ratio. If the amount of stabiliserexceeds the optimal amount, the excess does not directly interact withthe protein but rather forms particles of pure stabiliser. Thisbehaviour has been evidenced by Microscopy and by Differential ScanningCalorimetry (DSC) analysis.

The process for the co-precipitation of a substance with a stabiliser bya GAS process comprising the use of a supercritical fluid pure or mixedwith a modifier and a solution into a particle formation vessel may becarried out by the apparatus reported in Figure.

An advantage of the apparatus in FIG. 1 is related to the contactbetween supercritical fluid and solution since this takes place only inthe particle formation vessel. Hence any powder precipitation cannotoccur inside the nozzle and cause blockage. Importantly thesupercritical fluid acts as an anti-solvent but also promotes conversionof the solution into a fine spray as it enters the particle formationvessel. This widens the solution/anti-solvent interface and permits amore rapid mixing of the two phases and hence a rapid proteinprecipitation without any denaturation. In addition, the enhancement ofmass transfer rate between solution and supercritical fluid allowsoperation at mild temperature and pressure conditions which contributeto the avoidance of any possible protein denaturation. The apparatus ofFIG. 1 includes a particle formation vessel 22. This is a standardreaction vessel of an appropriate volume. The temperature in the vesselis maintained constant by means of a heating jacket 21. The pressure inthe vessel is controlled by means of a micro metering valve 25.

The temperature and pressure in the particle formation vessel aremeasured by means of a thermocouple 29 and a pressure transducer 30.

The particles formed are retained by filter 23. This is a stainlesssteel basket, whose bottom is made by a sintered stainless steel disk(0.5/inn). A second filter 24 (0.5/inn) is put at the vessel outlet.

The supercritical fluid is withdrawn from cylinder 3, it is condensed bycooler 4 and pumped by means of pump 8 to the particle formation vesselthrough line 34. Prior to entry into the particle formation vessel, thesupercritical fluid is heated to the desired temperature by means ofpre-heater 14 and heater 17. The pre-heater 14 also acts as pulsationdamper. The supercritical fluid is also filtered by means of filter 15(0.5/inn). Temperature and pressure of the supercritical fluid priorentry into the precipitation vessel are measured by means ofthermocouple 29 and pressure transducer 30, respectively.

The modifier is withdrawn from tank 2, it is pumped by means of pump 9to line 34 and it is mixed with the supercritical fluid prior to entryinto the particle formation vessel. The modifier is also filtered bymeans of filter 12 (0.5 ym).

Line 34 is equipped with a relief valve 16.

The solution is withdrawn from tank 1, it is pumped by means of pump 10to the particle formation vessel through line 36. The solution is alsofiltered by means of filter 13 (0.5 ym).

In another version of the process, the modifier may be introduced intothe particle formation vessel in part with the solution and in part withthe supercritical fluid. The supercritical fluid, pure or mixed with themodifier, and the solution are fed into the particle formation vessel bymeans of the nozzle 27.

Downstream the precipitation vessel 22, the mixture of supercriticalfluid, modifier and solvent are filtered by means of the filter 24 (0.5um) to retain the particles that are eventually not retained by filter23. The mixture of supercritical fluid, modifier and solvent isdepressurized by means of micro metering valve 25, the supercriticalsolvent is separated from the modifier and the solvent in the separator26, its flow rate is measured by means of mass flow meter 31 and it isdischarged.

The nozzle which is shown in FIGS. 2 and 3 allows introduction of thesolution and the supercritical fluid, pure or mixed with the modifier,into the particle formation vessel in co-current flow. The solution andsupercritical fluid velocities at the nozzle outlet are related to themass flow rate and to the diameter of orifices. Moreover, it ispreferred that the energy pressure of both solution and supercriticalfluid are converted into kinetic energy with a minimum energy loss. Thenozzle of FIGS. 2 and 3 was, in fact, designed for this purpose. Thepeculiarity of this nozzle is that the expansion of solution andsupercritical fluid occurs through orifices. An orifice is characterizedby a length to diameter ratio ranging from 5 to 10. It has the advantageover the capillary of minimizing the pressure energy loss and ofefficiently converting the pressure energy into kinetic energy. Thenozzle has orifices with diameters ranging from 0.02 to 0.04 mm andlength ranging from 0.1 to 0.2 mm. Such dimensions allow very highvelocities at the orifice outlet for both solution and supercriticalfluid.

The nozzle may be made of stainless steel, or of other appropriatematerial.

The nozzle is a disk with an orifice 39 at its centre and two or moreorifices 41 drilled at the same distance from the centre and evenlyspaced along a circumference. The orifices communicate with the interiorof the particle formation vessel. The solution is introduced into theparticle formation vessel through the central orifice, the supercriticalfluid, pure or with the modifier, is introduced into the particleformation vessel through the outer orifices. The solution 37 passesthrough a passage of diameter D3. Its end has a conical shape 40. At theapex of the conical end 40 there is a laser drilled orifice 39. Thelength L1 of the central orifice is 5 to 10 times its diameter D1. Thediameter D1 can be chosen in such a way to obtain any desired velocityof the solution at the orifice outlet.

The supercritical fluid 38 passes through passages of diameter D4. Eachpassage end has a conical shape 42. At the apex of the conical end 42there is a laser drilled orifice 41. The length 21 of the orifice is 5to 10 times its diameter D2. The diameter D2 can be chosen in such a wayto obtain any desired velocity of the supercritical fluid at the orificeoutlet.

The ratio between length (L1 or L2) and diameter (D1 or D2) of theorifices 39 and 41 are chosen so as to set to a minimum the energy lossand to obtain higher velocities by converting energy pressure intokinetic energy.

The solution emerges from the central orifice 39 at high velocity and itis broken in fine droplets coming in contact with the supercriticalfluid. The dispersion of the solution liquid jet is enhanced by thesupercritical fluid emerging from orifices 41, provided that thesupercritical fluid velocity is very high, of the order of magnitude ofthe velocity of sound at the working temperature and pressure. Theeffect of the supercritical fluid in enhancing the dispersion of thesolution liquid jet is crucial and determines the shape, size and yieldof the product.

Orifices can be drilled with diameters down to 0.02 mm. The nozzles thathave been used for carrying out the tests have orifices of diameterranging from 0.02 to 0.04 mm. In another embodiment of the invention,one or more of the outer orifices are drilled in such a way that theiraxes converge on the axis of the central orifice. The angle formed bythe axis of the outer orifices with the axis of the central orifice isbetween 1 and 30°.

A crucial point in the process for fine dry protein micro-particleformation is the mixing of the solution with the supercritical fluid: arapid and intimate mixing causes precipitation of particles with a smalldiameter and enables a high powder yield.

In order to have a good mixing, the solution should be dispersed intothe supercritical fluid in form of small droplets, thus providing highinterfacial area for mass transfer and a short path for the diffusion ofsupercritical fluid in the solution droplets and thereby preventing thegrowth of solute particles. Moreover a high ratio between flow rate ofsupercritical fluid and flow rate of solution causes a large excess ofthe supercritical fluid to solution at the moment of their contact,enhancing the driving force for mass transfer of the supercritical fluidinto the solution and of solvent into supercritical fluid.

When the solvent solubility in the supercritical fluid is low, the useof a modifier allows a better mixing between solution and supercriticalfluid.

When a modifier is used, the ratio of modifier flow rate and of solutionflow rate has to be chosen so that a high increase in solubility ofsolvent in the supercritical fluid is obtained. The modifier can beintroduced with the supercritical fluid or with the solution or in partwith the supercritical fluid and in part with the solution. The way ofintroduction of the modifier greatly influences the extraction of thesolvent and the structure of particles that are formed.

For the precipitation of powders from aqueous solution using carbondioxide as supercritical solvent and ethanol as modifier the ratiobetween supercritical fluid flow rate and the modifier flow rate ispreferably within the range 4-8, more preferably 7, while the ratiobetween modifier flow rate and the solution flow rate is preferablywithin the range 15-25 and more preferably 20.

As pointed out above, it is necessary to have a good dispersion of thesolution into the supercritical fluid in order to obtain very smalldroplets of solution.

The size of the formed solution droplets is determined by thefluidodynamic conditions in the mixing zone and by the physicalproperties of solution and supercritical solvent, such as viscosity,surface tension, density. These properties are greatly influenced by thetemperature and pressure of the supercritical fluid.

The supercritical fluid inlets are positioned around the solution inletand at a very short distance therefrom (about 3 mm): this configurationallows the solution to be energized by the supercritical fluid thusenhancing the dispersion of the solution into very fine droplets,providing high interfacial surface are between the two phases and fastextraction of solvent into supercritical fluid. These phenomena areparticularly efficient when the supercritical fluid velocity at theorifice outlet reaches or is greater than the speed of sound causing aMach disc formation and solution dispersion as very fine droplets(Matson D. W., Fulton J. L., Petersen R. C., Smith R. D., “Rapidexpansion of supercritical fluid solutions: solute formation of powders,thin films, and fibers” Ind. Eng. Chem. Res., 1987 26, 22982306). Thespeed of sound in a fluid is strongly dependent on pressure andtemperature: the minimum value of speed of sound for carbon dioxide inthe supercritical region is 208 m/s at 8 MPa and 40° C. To takeadvantage of the above mentioned phenomena it is convenient to workaround the value of speed of sound for carbon dioxide in thesupercritical region, e.g., 208 m/s at 8 MPa and 40° C.

For the production of fine powders from aqueous solutions with the GASprocess using carbon dioxide as supercritical solvent and ethanol asmodifier, it was found that optimal operative conditions are 8-12 MPaand 35-50° C. In the experimental apparatus used for carrying out theexperimental tests, the supercritical fluid mass flow rate was 30 g/min,the solution flow rate 0.2 g/min, and the modifier mass flow rate 4g/min, having set the ratio of supercritical fluid to modifier mass flowrate at 7 and the ratio of modifier to solution mass flow rate at 20 andsupercritical fluid velocity at the nozzle outlet at about 300 m/s.Using this apparatus we carried out the process to produce stable drymicro-particles of a substance and a stabiliser by GAS co-precipitation.Proteins such as Alkaline Phosphatase and Lysozyme were used as thesubstance and Trehalose as stabiliser. Co-precipitated powders atdifferent protein/stabiliser ratio were produced. The yield of thecollected powder was 90%. The retained enzymatic activity after theprocess was found to be within 95% and 100%, compared to the unprocessedcommercial reagent. The particle size distributions of these powdersshowed that more than 90% of the particles have an equivalent diameterless then 10 um with a narrow size distribution. Furthermore, thephysicochemical characterisation showed that co-precipitation allowsintimate interactions between protein and stabiliser molecules and foreach protein/stabiliser couple there is an optimal weight/weight ratio.Finally, stability studies showed that the Alkalinephosphatase/trehalose co-precipitated particles were more stable thanthe equivalent freeze-dried product.

EXPERIMENTAL PROCEDURE

The supercritical fluid is fed to the precipitation vessel by means ofpump 8, which is used to set the supercritical fluid flow rate. Thetemperature of the supercritical fluid in line 35 is set by means ofheater 17 to a higher value than the temperature inside the particleformation vessel, to take into account the temperature lowering due tothe expansion through the nozzle orifices. The modifier is then added ata predetermined flow rate to the supercritical fluid by means of pump 9.The solution of protein and stabiliser is pumped by means of pump 10into the particle formation vessel when steady state conditions areattained.

After that a certain amount of solution is fed into the particleformation vessel, pumps 9 and 10 are stopped and only the supercriticalfluid is fed to the particle formation vessel as long as theprecipitated powder is free of solvent and modifier.

The particle formation vessel is depressurized, the powder is recoveredand sealed in 10 mL vials under dry nitrogen.

The co-precipitated proteins stability was tested by storing vials atthe following conditions: 25° C.-60% RH; 30° C.-65% RH; 40° C.-75% RH.Each sample was analysed for biological activity at t=0, 1, 2, 3 and 6months. As comparison, a parallel study was conducted on proteinprecipitated by supercritical fluids as is, on analogous freeze-driedproducts and on the unprocessed commercial product, all stored under drynitrogen.

Example 1 Preparation of Alkaline Phosphatase (ALP)/TrehaloseCo-Precipitate Particles

In this example, the method of the invention is used to co-precipitatemixtures of alkaline phosphatase (ALP) and trehalose.

Solutions containing ALP (SIGMA Chemicals) at concentration 0.2% w/w andtrehalose (SIGMA Chemicals) at concentration within the range 0-2% w/win deionized water were used.

The ALP/Trehalose ratios of the obtained powders were as follow: 1:10,1:2 and 1:0. Carbon dioxide as supercritical fluid and ethanol asmodifier were used.

The solution was fed into the particle formation vessel 22 by means ofpump 10 at a flow rate of 0.2 g/min. Supercritical carbon dioxide wasfed by means of pump 8 at a flow rate of 30 g/min, ethanol was fed bymeans of pump 9 to line 34 at a flow rate of 4 g/min and it was mixedwith supercritical carbon dioxide prior to entry into the particleformation vessel.

The supercritical fluid was injected into the particle formation vesselthrough the four external orifices of the nozzle, each with a diameterof 0.04 mm. The solution was injected into the particle formation vesselthrough the central orifice of the nozzle, having a diameter of 0.04 mm.The length of all orifices was 0.2 mm.

Temperature and pressure inside the particle formation vessel weremaintained at a constant 40° C., by means of the heating jacket 21, and100±1 bar by means of the micro metering regulation valve 25,respectively. Precipitated particles were collected on the filter 23 atthe bottom of particle formation vessel, while supercritical fluid,modifier and water were collected into the cylinder 26 at atmosphericpressure.

The process was conducted as long as a sufficient amount of powder wasobtained. After the solution and modifier feeds were stopped and onlypure carbon dioxide was fed into particles formation vessel in order toextract any trace of solvent and modifier from the precipitated powders.Typically, the particle formation vessel was washed with two volumes ofcarbon dioxide in order to obtain dry powders.

After depressurization, the particle formation vessel was opened and thepowders were recovered and stored in 10 mL vials under dry nitrogen.

The yield of the collected powder was 90%.

The residual enzymatic activity of ALP was within 95% and 100%, comparedto the unprocessed commercial reagent. The powder optical microscopyanalysis shows that at high trehalose content as for ALP/trehalose ratio1:10 the powder is formed by two different population of particles: one,which is the largely frequent, is formed by needle shaped particles,while the other by round shaped particles. The needle shaped particlesare quite similar to those obtained by trehalose as is precipitation bysupercritical CO₂. The lower trehalose content powders show the roundshaped particles population only. Thus, the trehalose can beco-precipitated with ALP by supercritical CO₂ to give one kind ofparticles only at the lower trehalose contents (protein/trehalose ratios1:2). Similar behaviour was found for lysozyme/trehalose co-precipitates(see example 2). Analogous products were prepared by freeze-drying. Inthis case the found residual enzymatic activity of ALP was within 95%and 104%, compared to the unprocessed commercial reagent.

Similar vials containing unprocessed commercial ALP or analogousfreeze-dried products, all under dry nitrogen, were prepared.

Stability Study

Several vials of each category were placed at each of the followingconditions: 25° C.-60% RH; 30° C.-65% RH; 40° C.-75% RH for 6 months. Att=0, 1, 2, 3, 6 months, the contents of the vials were assayed for ALPactivity. The stability studies results are summarized in Table 1.

Pure ALP precipitated by supercritical CO₂ (sample F6) shows a decay ofenzymatic activity at all the conditions. The residual activity after 6months at 40° C.-75% RH (most extreme conditions), is 57% of the t=0value.

On the contrary, no significant loss of activity at all conditions up to6 months was found for ALP/trehalose co-precipitated by supercriticalCO₂ at ratio 1:10 (sample FT8).

At 40° C.-75% RH, pure freeze-dried ALP (sample F8) and SIGMA commercialproduct show similar decays and only 43% and 42% of the initialenzymatic activity was retained after 6 months. At the other conditions,instead, the SIGMA product shows a slower activity loss than the purefreeze-dried ALP. In fact after 6 months the following residualenzymatic activity were detected: 95% vs 83% at 25° C.-60% RH, and 86%vs 76% at 30° C.-65% RH.

Finally, the freeze dried powder having the ALP/trehalose ratio of 1:10(sample FT10) showed an initial rapid loss of activity, then a slowerone up to six months which seams to be independent to the storageconditions. In fact the retained enzymatic activities at 25° C.-60% RH,30° C.-65% RH and 40° C.-75% are 90%, 88% and 90% respectively of theinitial value.

Example 2 Preparation of Lysozyme/Trehalose Co-Precipitate Particles

In this example, the method of the invention is used to prepareco-precipitate powders using lysozyme and trehalose.

Solutions containing lysozyme (SIGMA Chemicals) at concentration within0.2-1% w/w and trehalose (SIGMA Chemicals) at concentration within therange 0-2% w/w in deionized water were used. The lysozyme/trehaloseratios of the obtained powders were as follows: 1:10, 1:5, 1:2, 1:1,2:1, 4:1 and 1:0 (Table 2).

Carbon dioxide was used as supercritical fluid and ethanol as modifier.

The aqueous solution containing the enzyme and the stabiliser was fedinto the particle formation vessel 22 by means of pump 10 at a flow rateof 0.2 g/min. Supercritical carbon dioxide was fed by means of pump 8 ata flow rate of 30 g/min, ethanol was fed by means of pump 9 to line 34at a flow rate of 4 g/min and it was mixed with supercritical carbondioxide before entering into the particle formation vessel.

The supercritical fluid was injected into the particle formation vesselthrough the four external orifices of the nozzle, each with a diameterof 0.04 mm. The solution was injected into the particle formation vesselthrough the central orifice of the nozzle, having a diameter of 0.04 mm.Length of all orifices is 0.2 mm.

Temperature and pressure inside the particle formation vessel weremaintained at a constant 40° C., by means of the heating jacket 21, and100±1 bar by means of the micro metering regulation valve 25,respectively.

Precipitated particles were collected on the filter 23 at the bottom ofparticle formation vessel, while supercritical fluid, modifier, waterand solute eventually not precipitated were collected in the cylinder 26at atmospheric pressure.

After that, a certain amount of solute was fed into the particleformation vessel, pumps 9 and 10 were stopped and only supercriticalfluid was fed into the particle formation vessel in order to dry theprecipitated powders: typically, it requires about two times the volumeof the particles formation vessel to obtain dry powders.

At this point, the particle formation vessel was depressurized, openedand the powders collected.

The yield of recovered powders was 90%.

The found residual enzymatic activity of lysozyme was within 96% and100%, compared to the unprocessed commercial reagent.

Table 2 reports for each sample the lysozyme/trehalose ratio, theretained enzymatic activity, the protein content which is related tohomogeneity of precipitation, the number of particles populations andthe particle sizes. As can be noted, for all the samples, both enzymaticactivity and protein content are very close to theoretical values. Thusthe experimental conditions we used allowed a similar precipitation forboth protein and sugar and guaranteed an almost complete biologicalactivity recovery.

The particle size distributions of powders calculated by image analysisof SEM micrographs showed that for all the powders obtained bysupercritical CO₂ precipitation, more than 90% of the particles have anequivalent diameter less then 10 um with a narrow size distribution.FIGS. 4, 5, 6 show the particle size distributions of supercritical CO₂co-precipitated with lysozyme/trehalose ratios 1:10, 1:2 and 1:0respectively. The other co-precipitates yielded similar distributions.Moreover, the powders observation by optical microscopy analysis showedthat at high trehalose contents as for both lysozyme/trehalose ratios1:10 and 1:5, the powders were composed of two particle populations: theone, which largely was the most frequent, was formed by needle shapedparticles, the other was formed by round shaped particles. The needleshaped particles were quite similar to those obtained by trehalose asprecipitated by supercritical CO₂. On the contrary, at the lowertrehalose content (lysozyme/trehalose ratio 1:2) the powders showed theround shaped particles population only. Thus lysozyme can beco-precipitated with trehalose by supercritical CO₂ to form only onekind of particle at the lower trehalose content (higherprotein/trehalose ratios). Hence an optimal value for protein/trehaloseratio which guarantees the best interaction between the two kind ofmolecules exists. This behaviour has been confirmed by DSC analysis.FIG. 7 shows DSC thermograms of various co-precipitated lysozymepowders. As reference, pure precipitated lysozyme and trehalose are alsoreported. As can be noted, at higher trehalose content (ratio 1:5),samples contain amorphous trehalose which recovers the crystalline abit(exothermic peak at 197° C.) and then melts at 214° C. in the same wayof precipitated trehalose itself. The thermal behaviour of lower contenttrehalose samples is quite different. The samples with ratios 1:2 to 4:1show thermograms similar to the one of lysozyme as is. The most relevantdifference is the shift towards lower temperatures of the characteristictransition of lysozyme at T=204° C. The higher the trehalose content thelower the temperature of transition. Thus, we have strong evidence thatco-precipitation by supercritical fluids allows an intimate interactionbetween protein and trehalose. In fact up to a defined amount of sugar(1:2 ratio) we obtained a homogeneous solid phase. This ratio is able toprovide the best protein/sugar interaction and the best long termstability of protein. TABLE 1 Enz. Enz. Enz. Enz. Sample/ ALP/ Activityat Activity at Activity at Activity at drying Trehalose Storage 1 month2 months 3 months 6 months method Ratio condition (% of t = 0) (% of t =0) (% of t = 0) (% of t = 0) ALP 1:0 −20° C. — — — 96 Sigma 25° C./60%RH 101  100  101  95 Freeze- 30° C./70% RH 103  103  94 86 drying 40°C./75% RH 94 85 75 42 ALP F6 1:0 −20° C. — — — — SCF 25° C./60% RH 91 7269 67 30° C./70% RH 101  71 59 59 40° C./75% RH 64 69 58 57 ALP F8 1:0−20° C. — — — — Freeze- 25° C./60% RH 88 89 89 83 drying 30° C./70% RH83 87 87 76 40° C./75% RH 75 75 62 43 ALP FT8  1:10 −20° C. — — — 101 SCF 25° C./60% RH 111  99 113  99 30° C./70% RH 104  97 102  99 40°C./75% RH 113  98 101  99 ALP FT10  1:10 −20° C. — — — 95 Freeze- 25°C./60% RH 95 95 93 90 drying 30° C./70% RH 94 96 92 88 40° C./75% RH 9493 92 90

TABLE 2 Lysozyme I Enz. Activity Protein No. of Trehalose (mg Enz. I mgcontent (% on particles Particles size SAMPLE Ratio Prot.) normal)populations (% <10 jim) L3 1:0 0.96 102.6 1 99 LT2  1:10 1.04 104.3 2 —LT3 1:1 0.96 100.8 1 98 LT6 1:5 0.96 104.0 2 92 LT8 4:1 1.01 103.2 1 97LT9 1:2 0.98 104.0 1 93 LT10 2:1 0.97 103.9 1 97

1. A coprecipitate of protein or polypeptide and stabiliser obtained bya gas anti solvent process comprising introducing into a particleformation vessel a supercritical fluid pure or mixed with a modifier;and a. a solution comprising said protein or polypeptide and saidstabiliser dissolved in a solvent; b. so as said solvent is extractedfrom the solution by said supercritical fluid and co-precipitation ofsaid protein or polypeptide and stabiliser occurs, wherein saidstabiliser is a sugar.
 2. A coprecipitate as claimed in claim 1 whereinthe solution comprises a protein and coprecipitation of the protein andstabiliser occurs.
 3. A coprecipitate according to claim 1 wherein saidsolution and said supercritical fluid are introduced into said particleformation vessel via separate inlet nozzles.
 4. A coprecipitateaccording to claim 3 wherein said supercritical fluid is introduced intosaid particle formation vessel via a plurality of inlet nozzles.
 5. Acoprecipitate as claimed in claim 4 wherein said nozzles are present ona disk, the solution inlet nozzle being in the centre of said disksurrounded by a plurality of supercritical fluid inlet nozzles evenlyspaced along the circumference thereof.
 6. A coprecipitate according toclaim 1, wherein the solution is introduced into the particle formationvessel mixed with a modifier.
 7. A coprecipitate according to claim 1,wherein the supercritical fluid is introduced into the particleformation vessel mixed with a modifier.
 8. A coprecipitate according toclaim 1, wherein said supercritical fluid is selected from carbondioxide, ethane, ethylene, propane, sulfur hexafluoride, nitrous oxide,chlorotrifluoromethane, monofluoromethane, xenon and mixtures thereof.9. A coprecipitate according to claim 1, wherein said solvent isselected from water, ethanol, methanol, DMSO, isopropanol, acetone, THF,acetic acid, ethylene glycol, polyethylene glycol, andN,N-dimethylaniline and mixtures thereof.
 10. A coprecipitate accordingto claim 1, wherein said modifier, which is different from said solvent,is selected from water, ethanol, methanol, DMSO, isopropanol, acetone,THF, acetic acid, ethylene glycol, polyethylene glycol, andN,N-dimethylaniline and mixtures thereof.
 11. A coprecipitate accordingto claim 1, wherein the supercritical fluid is carbon dioxide, thesolvent is water, and the modifier is ethanol.
 12. A coprecipitateaccording to claim 1, wherein the stabiliser is a trehalose.
 13. Acoprecipitate according to claim 1, wherein the ratio of protein orpolypeptide to stabiliser in the solution is 1:1 to 10 w/w.
 14. Acoprecipitate according to claim 13, wherein the ratio of protein orpolypeptide to stabiliser in the solution is 1:2 w/w.
 15. Acoprecipitate as claimed in claim 1, wherein the supercritical fluidenters the particle formation vessel at the speed of sound in the fluidor greater.
 16. A coprecipitate as claimed in claim 1, wherein amodifier is used and the ratio between fluid flow rate and modifier flowrate range 4:1 to 8:1 w/w. supercritical is within the
 17. Acoprecipitate as claimed in claim 1, wherein a modifier is used and theratio between modifier flow rate and solution flow rate is within therange 15:1 to 25:1 w/w.
 18. A coprecipitate according to claim 13,wherein the ratio of protein or polypeptide to stabiliser in thesolution is 1:2 to 4:1 w/w.